Cryo-EM studies of a metazoan replisome captured ex vivo during elongation and termination

Our bodies are built-up of trillions of cells. Over time, our cells age and become damaged, so a subset of cells in our bodies keep dividing, creating replacements. Before each cell division, every cell must first duplicate its genome - all of it, just once and without mistakes. Mistakes during DNA replication, which are not timely repaired, can lead to mutations and genetic changes that in turn can lead to problems with cell proliferation, aging, and development of cancer. Most of the cancer-driving mutations result from random mistakes during the process of DNA replication. Moreover, hereditary mutations in components of the DNA replication machinery cause a set of disorders characterised by small posture and small brain due to the inability to create enough cells to develop a normal-sized human being.
Replicating all of our DNA is a huge task - we have about 2 metres of DNA in each of our cells, and it is compacted in a highly organised way to fit into the nucleus in a manner that enables proteins to access any needed DNA sequences. During DNA replication this structure must be unwound, duplicated, and compacted again. To replicate all DNA, the process of DNA replication starts from about 50 thousand start sites with about 100 thousand individual replication machineries (replisomes) replicating DNA. Ever since Watson and Crick proposed the first model of DNA replication 70 years ago, researchers aim to understand how this process is coordinated, regulated, and delivered without mistakes.

In eukaryotic cells, the replication machinery is composed of hundreds of proteins that must be precisely organised to coordinate all their functions together. Our previous work has shown that the core of the replisome is organised around the replicative helicase (CMG complex). The replicative helicase can unwind double-stranded DNA to provide the template for synthesis of the complementary strands. Over the last 15 years, structural biology findings have produced the first structures of reconstituted helicase providing a great breakthrough into our understanding of how some of the components of the replication machinery are working together. However, almost all the solved complexes were assembled in vitro from purified proteins. This approach is obviously very successful, but it requires pre-determined known factors that are assumed to form the complex of interest, potentially missing additional or minor partners that could affect the overall structure of the complex. Moreover, the molecular machineries involved in these processes are naturally assembled on a chromatinised substrate and are tightly regulated. Since reconstituted complexes are assembled in vitro, elements of that regulation are missing, thus potentially leading to incomplete or misleading observations. Finally, most of the solved structures are reconstituted from budding yeast proteins, which are not identical to proteins from human or other higher eukaryotic organisms.
We propose here to optimize an alternative method to isolate protein complexes essential for DNA replication using Xenopus laevis egg extract, which is the only higher eukaryote cell-free system containing all the factors involved in DNA replication. The purified protein complexes will be analysed via structural microscopy techniques and biochemical approaches delivering the first ever naturally (ex vivo) assembled structures of a replicative helicase and the replisome. We will biochemically validate our structures and compare them to the existing in vitro assembled structures from other species. Moreover, using our expertise of working with this system, we can use various inhibitors to "freeze" the replication machinery in various configurations: active, stalled, terminated. We will solve their structures and compare them, to understand the dynamic changes that occur to the replisome as it transitions through these states.

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing but also neuropathology and cancer development in humans. The protein machinery delivering this gargantuan task is formed from hundreds of proteins organised in several subcomplexes, with the core replisome built around the replicative helicase at the tip of the DNA replication fork.
Over the last 15 years, biochemical and structural protein work has provided a much better understanding of how the replicative helicase is assembled during replication origin licensing and initiation and has transformed our understanding of these stages of DNA replication. The first steps have also begun in solving structures of the terminated replicative helicases, providing a breakthrough in understanding the selectivity of the unloading process. However, most of these structures have been solved using budding yeast complexes and almost all consist of complexes assembled in vitro from purified proteins. The molecular machineries involved in DNA replication are naturally assembled onto chromatinised substrate in a tightly regulated manner. Since reconstituted complexes are assembled in vitro, elements of that regulation are likely missing, thus potentially leading to incomplete observations. We propose to optimize an alternative method, to isolate protein complexes essential for DNA replication using Xenopus laevis egg extract, which is the only higher eukaryotic system possessing cell cycle and chromatin-regulated DNA replication activity. We will purify replisome complexes enriched at specific stages of their activity: active, stalled or terminated replisomes. The purified complexes will be analysed via cryo-EM and cross-linking mass spectrometry (XL-MS) delivering the first ever naturally (ex vivo) assembled structure of the replicative helicase and the replisome.